Preexisting maternal immunity to AAV but not Cas9 impairs in utero gene editing in mice.

In utero gene editing (IUGE) is a potential treatment for inherited diseases that cause pathology before or soon after birth. Preexisting immunity to adeno-associated virus (AAV) vectors and Cas9 endonuclease may limit postnatal gene editing. The tolerogenic fetal immune system minimizes a fetal immune barrier to IUGE. However, the ability of maternal immunity to limit fetal gene editing remains a question. We investigated whether preexisting maternal immunity to AAV or Cas9 impairs IUGE. Using a combination of fluorescent reporter mice and a murine model of a metabolic liver disease, we demonstrated that maternal anti-AAV IgG antibodies were efficiently transferred from dam to fetus and impaired IUGE in a maternal titer-dependent fashion. By contrast, maternal cellular immunity was inefficiently transferred to the fetus, and neither maternal cellular nor humoral immunity to Cas9 impaired IUGE. Using human umbilical cord and maternal blood samples collected from mid- to late-gestation pregnancies, we demonstrated that maternal-fetal transmission of anti-AAV IgG was inefficient in midgestation compared with term, suggesting that the maternal immune barrier to clinical IUGE would be less relevant at midgestation. These findings support immunologic advantages for IUGE and inform maternal preprocedural testing protocols and exclusion criteria for future clinical trials.

CT imaging-derived phenotypes for abdominal muscle and their association with age and sex in a medical biobank.

The study of muscle mass as an imaging-derived phenotype (IDP) may yield new insights into determining the normal and pathologic variations in muscle mass in the population. This can be done by determining 3D abdominal muscle mass from 12 distinct abdominal muscle regions and groups using computed tomography (CT) in a racially diverse medical biobank. To develop a fully automatic technique for assessment of CT abdominal muscle IDPs and preliminarily determine abdominal muscle IDP variations with age and sex in a clinically and racially diverse medical biobank. This retrospective study was conducted using the Penn Medicine BioBank (PMBB), a research protocol that recruits adult participants during outpatient visits at hospitals in the Penn Medicine network. We developed a deep residual U-Net (ResUNet) to segment 12 abdominal muscle groups including the left and right psoas, quadratus lumborum, erector spinae, gluteus medius, rectus abdominis, and lateral abdominals. 110 CT studies were randomly selected for training, validation, and testing. 44 of the 110 CT studies were selected to enrich the dataset with representative cases of intra-abdominal and abdominal wall pathology. The studies were divided into non-overlapping training, validation and testing sets. Model performance was evaluated using the Sørensen-Dice coefficient. Volumes of individual muscle groups were plotted to distribution curves. To investigate associations between muscle IDPs, age, and sex, deep learning model segmentations were performed on a larger abdominal CT dataset from PMBB consisting of 295 studies. Multivariable models were used to determine relationships between muscle mass, age and sex. The model's performance (Dice scores) on the test data was the following: psoas: 0.85 ± 0.12, quadratus lumborum: 0.72 ± 0.14, erector spinae: 0.92 ± 0.07, gluteus medius: 0.90 ± 0.08, rectus abdominis: 0.85 ± 0.08, lateral abdominals: 0.85 ± 0.09. The average Dice score across all muscle groups was 0.86 ± 0.11. Average total muscle mass for females was 2041 ± 560.7 g with a high of 2256 ± 560.1 g (41-50 year old cohort) and a change of - 0.96 g/year, declining to an average mass of 1579 ± 408.8 g (81-100 year old cohort). Average total muscle mass for males was 3086 ± 769.1 g with a high of 3385 ± 819.3 g (51-60 year old cohort) and a change of - 1.73 g/year, declining to an average mass of 2629 ± 536.7 g (81-100 year old cohort). Quadratus lumborum was most highly correlated with age for both sexes (correlation coefficient of - 0.5). Gluteus medius mass in females was positively correlated with age with a coefficient of 0.22. These preliminary findings show that our CNN can automate detailed abdominal muscle volume measurement. Unlike prior efforts, this technique provides 3D muscle segmentations of individual muscles. This technique will dramatically impact sarcopenia diagnosis and research, elucidating its clinical and public health implications. Our results suggest a peak age range for muscle mass and an expected rate of decline, both of which vary between genders. Future goals are to investigate genetic variants for sarcopenia and malnutrition, while describing genotype-phenotype associations of muscle mass in healthy humans using imaging-derived phenotypes. It is feasible to obtain 3D abdominal muscle IDPs with high accuracy from patients in a medical biobank using fully automated machine learning methods. Abdominal muscle IDPs showed significant variations in lean mass by age and sex. In the future, this tool can be leveraged to perform a genome-wide association study across the medical biobank and determine genetic variants associated with early or accelerated muscle wasting.

Immune Modulation Permits Tolerance and Engraftment in a Murine Model of Late-Gestation Transplantation.

In utero hematopoietic cell transplantation (IUHCT) is an experimental non-myeloablative therapy with potential application to hematologic disorders including Sickle cell disease. Its clinical utility has been limited due to the early acquisition of T cell immunity beginning at approximately 14 weeks gestation, posing significant technical challenges and excluding from treatment fetuses evaluated after the first trimester. Using murine neonatal transplantation at 20 days post-coitum (DPC) as a model for late-gestation transplantation (LGT) in humans, we investigated whether immune modulation with anti-CD3 monoclonal antibody (mAb) could achieve donor-specific tolerance and sustained allogeneic engraftment comparable to the early-gestation fetal recipient at 14 DPC. In allogeneic wild-type strain combinations, administration of anti-CD3 mAb with transplantation resulted in transient T cell depletion followed by central tolerance induction confirmed by donor-specific clonal deletion and skin graft tolerance. Normal immune responses to third-party major histocompatibility complex and viral pathogens were preserved, and graft-versus-host disease did not occur. We further demonstrate successful application of this approach to the Townes mouse model of Sickle cell disease. These findings confirm the developing fetal T cell response as a barrier to LGT and support transient T cell depletion as a safe and effective immunomodulatory strategy by which to overcome it.

Ionizable Lipid Nanoparticles for Therapeutic Base Editing of Congenital Brain Disease.

Delivery of mRNA-based therapeutics to the perinatal brain holds great potential in treating congenital brain diseases. However, nonviral delivery platforms that facilitate nucleic acid delivery in this environment have yet to be rigorously studied. Here, we screen a diverse library of ionizable lipid nanoparticles (LNPs) via intracerebroventricular (ICV) injection in both fetal and neonatal mice and identify an LNP formulation with greater functional mRNA delivery in the perinatal brain than an FDA-approved industry standard LNP. Following in vitro optimization of the top-performing LNP (C3 LNP) for codelivery of an adenine base editing platform, we improve the biochemical phenotype of a lysosomal storage disease in the neonatal mouse brain, exhibit proof-of-principle mRNA brain transfection in vivo in a fetal nonhuman primate model, and demonstrate the translational potential of C3 LNPs ex vivo in human patient-derived brain tissues. These LNPs may provide a clinically translatable platform for in utero and postnatal mRNA therapies including gene editing in the brain.

Molecular and Cellular In Utero Therapy.

Significant advances in maternal-fetal medicine and gene sequencing technology have fostered a new frontier of in utero molecular and cellular therapeutics, including gene editing, enzyme replacement therapy, and stem cell transplantation to treat single-gene disorders with limited postnatal treatment strategies. In utero therapies take advantage of unique developmental properties of the fetus to allow for the correction of monogenic disorders before irreversible disease pathology develops. While early preclinical studies in animal models are encouraging, more studies are needed to further evaluate their safety and efficacy prior to widespread clinical use.

Pulling Back from the Brink: A Multi-Pronged Approach to Address General Surgery Resident Clinical Work Hour Adherence.

PURPOSE: The conflict between prioritizing education for surgical trainees, promoting trainee wellness, and maintaining optimal patient care has remained challenging since the introduction of the Accreditation Council for Graduate Medical Education (ACGME) work hour restrictions in 2003. There is still a dearth of research examining which interventions successfully enable duty hour adherence. This study assessed the impact of a combination of strategic interventions on improving clinical work hour adherence. METHODS: Monthly clinical work hour submission rates were assessed for all general surgery residents at a single university-based residency program over a 3-year period (2018-2021). Interventions targeted 3 domains and were implemented between academic years 2018 to 2019 (control) and 2020 to 2021 (intervention): 1) improving the accuracy and transparency of work hour reporting, 2) facilitating more timely interventions, and 3) structural scheduling changes. All 80-hour work week and continuous work hour violations were assessed. Findings were also compared to the corresponding ACGME Resident Survey results. RESULTS: There was no significant difference in the rate of monthly work hour submissions pre- and postintervention (78% vs 75%, p = 0.057). However, the number of total reported monthly violations decreased significantly (mean 13.8 vs 2.4, p < 0.01), including decreases in both 80-hour work week and continuous work hour violations (mean 4.7 vs 1.6, p < 0.001 and 9.1 vs 0.8, p < 0.001, respectively). Reported compliance also increased on the annual ACGME resident surveys, where 61% vs 95% of residents felt they were compliant with the 80-hour work week and 71% vs 95% felt they were compliant with the continuous work hours (2018-19 vs 2020-21). CONCLUSION: Innovative strategies addressing schedule changes, the culture of work hour reporting, and early intervention significantly decreased the number of duty hour violations at our institution. Reported resident compliance also improved based on ACGME Resident Survey data. These data may inform similar multifaceted approaches at other institutions to improve overall work hour adherence.

Surface conjugation of antibodies improves nanoparticle uptake in bronchial epithelial cells.

BACKGROUND: Advances in Molecular Therapy have made gene editing through systemic or topical administration of reagents a feasible strategy to treat genetic diseases in a rational manner. Encapsulation of therapeutic agents in nanoparticles can improve intracellular delivery of therapeutic agents, provided that the nanoparticles are efficiently taken up within the target cells. In prior work we had established proof-of-principle that nanoparticles carrying gene editing reagents can mediate site-specific gene editing in fetal and adult animals in vivo that results in functional disease improvement in rodent models of ß-thalassemia and cystic fibrosis. Modification of the surface of nanoparticles to include targeting molecules (e.g. antibodies) holds the promise of improving cellular uptake and specific cellular binding. METHODS AND FINDINGS: To improve particle uptake for diseases of the airway, like cystic fibrosis, our group tested the impact of nanoparticle surface modification with cell surface marker antibodies on uptake in human bronchial epithelial cells in vitro. Binding kinetics of antibodies (Podoplanin, Muc 1, Surfactant Protein C, and Intracellular Adhesion Molecule-1 (ICAM)) were determined to select appropriate antibodies for cellular targeting. The best target-specific antibody among those screened was ICAM antibody. Surface conjugation of nanoparticles with antibodies against ICAM improved cellular uptake in bronchial epithelial cells up to 24-fold. CONCLUSIONS: This is a first demonstration of improved nanoparticle uptake in epithelial cells using conjugation of target specific antibodies. Improved binding, uptake or specificity of particles delivered systemically or to the luminal surface of the airway would potentially improve efficacy, reduce the necessary dose and thus safety of administered therapeutic agents. Incremental improvement in the efficacy and safety of particle-based therapeutic strategies may allow genetic diseases such as cystic fibrosis to be cured on a fundamental genetic level before birth or shortly after birth.

Settling the Score: Injury Severity Score Fails to Capture Nuances in Pediatric Trauma.

OBJECTIVES: Recent work has questioned the accuracy of the Injury Severity Score (ISS) and the Abbreviated Injury Scale (AIS) in the pediatric population. We sought to determine mortality rates in pediatric trauma patients at ISSs considered "severe" in adults and whether mortality would vary substantially between adults and children sustaining injuries with the same AIS. METHODS: Univariate logistic regression was used to generate mortality rates associated with ISS scores, for children (<16 years of age) and adults, using the 2016 National Trauma Data Bank. Mortality rates at an ISS of 15 were calculated in both groups. We similarly calculated ISS scores associated with mortality rates of 10%, 25%, and 50%. Receiver operating characteristic curves were constructed to compare the discriminative ability of ISS to predict mortality after blunt and penetrating injuries in adults and children. Mortality rates associated with 1 or more AIS 3 injuries per body region were defined. RESULTS: There were 855,454 cases, 86,414 (10.1%) of which were children. The ISS associated with 10%, 25%, and 50% mortality were 35, 44, and 53, respectively, in children; they were 27, 38, and 48 in adults. At an ISS of 15, pediatric mortality was 1.0%; in adults, it was 3.1%. A 3.1% mortality rate was not observed in children until an ISS of 25. On receiver operating characteristic analysis, the ISS performed better in children compared with adults (area under the curve, 0.965 vs 0.860 [P < 0.001]). Adults consistently suffered from higher mortality rates than did children with the same number of severe injuries to a body region, and mortality varied widely between specific selected AIS 3 injuries. CONCLUSIONS: Although the ISS predicts mortality well, children have lower mortality than do adults for the same ISS, and therefore, the accepted definition of severe injury is not equivalent between these 2 cohorts. Mortality risk is highly dependent on the specific nature of the injury, with large variability in outcomes despite identical AIS scores.

Pneumatosis intestinalis in children beyond the neonatal period: is it always benign?

PURPOSE: The significance and management of pediatric pneumatosis intestinalis (PI) remains poorly defined. We sought to add clarity in children beyond the neonatal period. METHODS: Pediatric patients 3 months-18 years admitted to a quaternary children's hospital with a diagnosis of PI were included in this retrospective study. Pathologic PI was defined as irreversible, transmural intestinal ischemia. RESULTS: 167 children were identified with PI. Of these children, 155 (92.8%) had benign PI and 12 (7.2%) developed pathologic PI. The most common underlying diagnosis for pathologic PI was global developmental delay (75%), although we identified a spectrum of underlying diagnoses at risk for PI. Physical exam notable for abdominal distension (p = 0.023) or guarding (p = 0.028), and imaging with portal venous gas (p < 0.001) or bowel distension (p = 0.001) were significantly associated with pathologic PI. Only 6.6% of all children underwent an operation. For those undergoing non-surgical management of benign PI, 75% of children received antibiotics and average duration of bowel rest was 6.8 days. CONCLUSIONS: PI in children is primarily a benign phenomenon and often does not warrant surgical intervention. Bowel rest and antibiotics are therapeutic strategies frequently used in the treatment of this finding.

Smart-phone Based Geofencing: A Novel Approach to Monitoring Clinical Work Hours in Surgery Residency.

OBJECTIVE: The Accreditation Council for Graduate Medical Education specifies strict requirements for clinical work hours during residency training, with serious consequences for violations. Self-reporting of work hours by trainees can be inaccurate due to recall bias, giving program directors limited data to influence change. We aimed to assess the impact of a smart-phone based geofencing application on submission rates for work hours and reported violations in a general surgery residency program at a university-based medical center. We also examined resident perceptions surrounding implementation and use of the application. METHODS: We compared clinical work hours submitted and violations reported during the pilot period (October-November 2019) with the months prior to the launch of the application (July-August 2019). PGY1 and PGY2 residents were eligible to use the application during and after this pilot period. Semi-structured interviews were used to assess resident perceptions. A retrospective review was conducted to compare reporting during the same time period from the prior academic year (2018-2019) for historical reference. Paired t-tests were used to analyze the data. RESULTS: Twenty-six residents (15 PGY1, 11 PGY2) were eligible for the intervention and 23 residents (88%) used the application. The mean number of violations reported decreased significantly during the pilot period compared with the months prior to the intervention (4.5 vs. 11, p = 0.04). The total rate of submissions was not significantly different after the intervention (85% vs. 82%, p = 0.42). The PGY1 mean submission rate decreased during the pilot period (91%-75%, p = 0.21) while the PGY2 submission rate increased (77%-91%, p = 0.07). Compared with historical data, there was an increase in overall total submission rates between academic years 2018/2019 and 2019/2020 (74% vs. 79%, p = 0.047) and an associated decrease in the mean number of monthly violations (14 vs. 6.25, p = 0.004). Thirteen (50%) residents (8 PGY1, 5 PGY2) volunteered for semi-structured interviews. Most participants found the application useful for recording and reporting clinical work hours. They noted an ease in the administrative burden as well as more accurate reporting associated with automated logging. Use of the application was not perceived to limit engagement with patient care; however, there were privacy concerns and some technical barriers were identified. The messaging regarding the application's use was identified as critical for implementation. CONCLUSIONS: The "real-time" data provided by a geofencing application in our program helped to reduce the number of work-hour violations reported and did not diminish resident engagement with patient care. Decreasing the administrative burden of recording work hours coupled with improving transparency and accuracy of submissions may be important mechanisms.

Mortality After Adolescent Firearm Injury: Effect of Trauma Center Designation.

PURPOSE: To determine the incidence and outcomes of firearm injuries in adolescents and the effect of trauma center (TC) designation on their mortality. METHODS: The National Trauma Data Bank (2010-2016) was queried for all encounters involving adolescents aged 13-16 years with firearm injuries. Multivariable logistic regression was employed to determine the association of covariates with mortality (α = .05). Propensity score matching was also used to explore the relationship between TC designation and mortality. RESULTS: A total of 9,029 adolescents met inclusion criteria. Patients aged 15 and 16 years compromised 77.8% of the cohort and were more often male (87.9% vs. 80.6%, p < .001), black (63.8% vs. 56.1%, p < .001), injured in the abdomen (25.4% vs. 22.4%, p = .007) or extremities (62.3% vs. 56.7%, p < .001), and incurred severe injuries (54.5% vs. 50.9%, p = .004) versus 13- and 14-year-old patients. Younger patients were more often injured in the head/neck (23.8% vs. 20.5%, p = .001). Multivariable logistic regression demonstrated no difference in mortality between age groups. Poor neurologic presentation, severe injury, abdominal, chest, and head injuries were all associated with an increased odds of death. Odds of mortality were 2.88 times higher at adult TCs compared to pediatric TCs (CI: 1.55-5.36, p = .001). However, using a 1:1 propensity score matching model, no difference in mortality was found between TC types (p = NS). CONCLUSIONS: Variability exists in outcomes for adolescents after firearm injuries. Understanding and identifying the potential differences between pediatric and adult TCs managing adolescent firearm victims may improve survival in all treatment venues, but these data support patients being treated at the closest available TC.

COVID-19 Pandemic Significantly Decreases Acute Surgical Complaints.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic significantly reduced elective surgery in the United States, but the impact of COVID-19 on acute surgical complaints and acute care surgery is unknown. STUDY DESIGN: A retrospective review was performed of all surgical consults at the Hospital of the University of Pennsylvania in the 30 days prior to and 30 days following confirmation of the first COVID-19 patient at the institution. Consults to all divisions within general surgery were included. RESULTS: Total surgical consult volume decreased by 43% in the post-COVID-19 period, with a significant reduction in the median daily consult volume from 14 to 8 (P < .0001). Changes in consult volume by patient location, chief complaint, and surgical division were variable, in aggregate reflecting a disproportionate decrease among less acute surgical complaints. The percentage of consults resulting in surgical intervention remained equal in the 2 periods (31% vs 28%, odds ratio 0.85, 95% CI 0.61-1.21, P = .38) with most but not all operation types decreasing in frequency. The rise in the COVID-19 inpatient census led to increased consultation for vascular access, accommodated at our center by the creation of a new surgical procedures team. CONCLUSION: The COVID-19 pandemic significantly altered the landscape of acute surgical complaints at our large academic hospital. An appreciation of these trends may be helpful to other Departments of Surgery around the country as they deploy staff and allocate resources in the COVID-19 era.

Alginate microparticles loaded with basic fibroblast growth factor induce tissue coverage in a rat model of myelomeningocele.

BACKGROUND/PURPOSE: We sought to develop a minimally invasive intra-amniotic therapy for prenatal treatment of myelomeningocele (MMC) in an established rat model. METHODS: Time-dated pregnant rats were gavage-fed retinoic acid to induce MMC. Groups received intraamniotic injections at E17.5 with alginate particles loaded with fluorescent dye, basic fibroblast growth factor (Alg-HSA-bFGF), fluorescently tagged albumin (Alginate-BSA-TR), free bFGF, blank alginate particles (Alg-Blank), or PBS. Groups were analyzed at 3 h for specific particle binding or at term (E21) to determine MMC coverage. RESULTS: Alginate microparticles demonstrated robust binding to the MMC defect 3 h after injection. Of those specimens analyzed at E21, 150 of 239 fetuses (62.8%) were viable. Moreover, 18 of 61 (30%) treated with Alg-HSA-bFGF showed evidence of soft tissue coverage compared to 0 of 24 noninjected (P = 0.0021), 0 of 13 PBS (P = 0.0297), and 0 of 42 free bFGF (P = P < 0.0001). Scaffolds of aggregated particles associated with disordered keratinized tissue were observed covering the defect in 2 of 18 (11%) Alg-BSA-TR and 3 of 19 (16%) Alg-Blank specimens. CONCLUSIONS: Injection of microparticles loaded with bFGF resulted in significant soft tissue coverage of the MMC defect compared to controls. Alginate microparticles without growth factors might result in scaffold development over the fetal MMC. TYPE OF STUDY: Basic science. LEVEL OF EVIDENCE: N/A.

A somatic activating NRAS variant associated with kaposiform lymphangiomatosis.

PURPOSE: Kaposiform lymphangiomatosis (KLA) is a rare, frequently aggressive, systemic disorder of the lymphatic vasculature, occurring primarily in children. Even with multimodal treatments, KLA has a poor prognosis and high mortality rate secondary to coagulopathy, effusions, and systemic involvement. We hypothesized that, as has recently been found for other vascular anomalies, KLA may be caused by somatic mosaic variants affecting vascular development. METHODS: We performed exome sequencing of tumor samples from five individuals with KLA, along with samples from uninvolved control tissue in three of the five. We used digital polymerase chain reaction (dPCR) to validate the exome findings and to screen KLA samples from six other individuals. RESULTS: We identified a somatic activating NRAS variant (c.182 A>G, p.Q61R) in lesional tissue from 10/11 individuals, at levels ranging from 1% to 28%, that was absent from the tested control tissues. CONCLUSION: The activating NRAS p.Q61R variant is a known "hotspot" variant, frequently identified in several types of human cancer, especially melanoma. KLA, therefore, joins a growing group of vascular malformations and tumors caused by somatic activating variants in the RAS/PI3K/mTOR signaling pathways. This discovery will expand treatment options for these high-risk patients as there is potential for use of targeted RAS pathway inhibitors.

Prophylactic Ureteral Stenting in Laparoscopic Colectomy: Revisiting Traditional Practice.

BACKGROUND: Prophylactic placement of ureteral stents is performed during open colectomy to aid in ureteral identification and to enhance detection of injury. The effects of this practice in laparoscopic colectomy are unknown. This study compares outcomes of patients undergoing laparoscopic colectomy with and without prophylactic ureteral stenting. METHODS: A retrospective cohort study at a tertiary academic medical center was performed. The primary outcome measure was the incidence of ureteral injury. Secondary outcomes evaluated included mortality, length of stay, procedural duration, and new-onset urinary complication (hematuria, dysuria, and urinary tract infection). RESULTS: In 702 consecutive patients undergoing elective laparoscopic colectomy from 2013 to 2016, prophylactic stents were placed in 261 (37%) patients. Two ureteral injuries occurred (0.3%), both in patients who underwent ureteral stent placement (P = 0.07) and were found and repaired intraoperatively. There was no in-hospital mortality. When accounting for age-adjusted Charlson comorbidity score, procedural indication, gender, BMI, and extent of resection, no difference in hospital length of stay (P = 0.79) was noted comparing patients with and without stenting. However, stent placement prolonged operating time (P = 0.03) and increased the risk of new-onset urinary complications (P = 0.04). CONCLUSIONS: In this study, ureteral injuries only occurred in those with stent placement. Prophylactic ureteral stents in laparoscopic colectomy are associated with increased operative time and urologic morbidity. Owing to the low prevalence of ureteral injury in the elective setting and the increased risk of urinary complications, use of prophylactic ureteral stenting should be highly selective.

In utero nanoparticle delivery for site-specific genome editing.

Genetic diseases can be diagnosed early during pregnancy, but many monogenic disorders continue to cause considerable neonatal and pediatric morbidity and mortality. Early intervention through intrauterine gene editing, however, could correct the genetic defect, potentially allowing for normal organ development, functional disease improvement, or cure. Here we demonstrate safe intravenous and intra-amniotic administration of polymeric nanoparticles to fetal mouse tissues at selected gestational ages with no effect on survival or postnatal growth. In utero introduction of nanoparticles containing peptide nucleic acids (PNAs) and donor DNAs corrects a disease-causing mutation in the ß-globin gene in a mouse model of human ß-thalassemia, yielding sustained postnatal elevation of blood hemoglobin levels into the normal range, reduced reticulocyte counts, reversal of splenomegaly, and improved survival, with no detected off-target mutations in partially homologous loci. This work may provide the basis for a safe and versatile method of fetal gene editing for human monogenic disorders.